Clemson University TigerPrints All Dissertations Dissertations 5-2013 CORRELATION OF PHYSICOCHEMICAL PROPERTIES OF CHONDROITIN SULFATE TO ITS IN-VITRO ABSORPTION AND ANTI- INFLAMMATORY ACTIVITY Lahari Surapaneni Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Part of the Food Science Commons Recommended Citation Surapaneni, Lahari, "CORRELATION OF PHYSICOCHEMICAL PROPERTIES OF CHONDROITIN SULFATE TO ITS IN- VITRO ABSORPTION AND ANTI-INFLAMMATORY ACTIVITY" (2013). All Dissertations. 1101. https://tigerprints.clemson.edu/all_dissertations/1101 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. CORRELATION OF PHYSICOCHEMICAL PROPERTIES OF CHONDROITIN SULFATE TO ITS IN-VITRO ABSORPTION AND ANTI-INFLAMMATORY ACTIVITY A Dissertation Presented to the Graduate School of Clemson University In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Food, Nutrition and Packaging Sciences by Lahari Surapaneni May 2013 Accepted by: Dr. Vivian Haley-Zitlin, Committee Chair Dr. Ashby B. Bodine, Committee Co-Chair Dr. Xiuping Jiang, Dr. James R. Brooks ABSTRACT The purpose of this study was to investigate if the molecular weight and degree of sulfation of chondroitin sulfate (CS) has an impact on its in vitro absorption and anti- inflammatory activity. For absorption studies, Caco-2 cells were incubated with eight CS samples of differing molecular weights (7 kDa - 35 kDa). The amount of CS transported into the basolateral side of the Caco-2 monolayer was quantitatively determined to calculate the permeability coefficients (Peff). The permeability coefficients of the eight different CS samples across Caco-2 cell monolayers were assessed. For anti-inflammatory studies, RAW 264.7 murine macrophage cells were pre incubated with the CS samples for an hour followed by addition of bacterial lipopolysaccharide (LPS). The anti-inflammatory activity of CS samples was measured by the ability of CS samples to inhibit expression of a panel of inflammatory cytokines- tumor necrosis factor-alpha (TNF-α), Interleukin-1 beta (IL-1β) and Interleukin-6 (IL-6). These inflammatory markers were quantitatively measured using ELISA and inhibition of nitric oxide (NO) production was measured using Griess reagent assay. -6 -1 Of the eight samples evaluated, four had a Peff value of 15 x 10 cm sec or higher indicating moderate to high absorption. Two of the four samples with higher Peff values were high molecular weight compounds (~ 35 kDa). At concentrations of 5 µg/ml as well as 15 µg/ml, CS samples significantly inhibited expression of LPS induced TNF-α. Expression of IL-6 was inhibited by some of the CS samples at 15 µg/ml concentration ii but not at 5µg/ml. Under the experimental conditions, IL-1β and NO were not useful in estimating the anti-inflammatory activity of the CS samples. Statistical analysis which examined the relation between molecular weight and each of these inflammatory markers revealed no correlation (p<0.05). Within the CS molecular weight range used in this experiment, the absorption of CS samples did not have a correlation with their molecular weights but, interestingly, correlation was observed between the absorption and percentage of 6-sulfated disaccharide in the CS samples. CS samples used in this study appeared to inhibit some of the inflammatory cytokines but no correlation seemed to exist between the molecular weights and anti-inflammatory activity of these samples. iii DEDICATION This work is dedicated to all my teachers and mentors, who have encouraged and inspired me to think deeper. iv ACKNOWLEDGMENTS I would like to thank my advisers, Dr. Bodine and Dr. Haley, for their endless support and guidance. Their confidence in me and vision has helped me become a better researcher. I have learnt a lot from them and they continue to inspire me. Some of the work done in this dissertation would not have been possible without the co-operation of Dr. James R. Brooks, Pharmavite LLC. I would also like to thank Dr. Xiuping Jiang for agreeing to serve on my committee and guide me throughout my Ph.D. I would like to express my special thanks to the research and development team at Pharmavite who provided me invaluable guidance in completing this project. I am very grateful to Dr. Tzeng and Dr. Huang for their help with the anti-inflammatory part of this study. Last but not least, I would like to thank my family for their unconditional support and unwavering faith in me. I thank all my good friends who have accompanied me on the road to a doctoral degree, and whose friendship will be treasured. My special thanks to Ramakrishna Podila and Pooja Puneet for their constant support and encouragement. v TABLE OF CONTENTS Page TITLE PAGE .................................................................................................................... i ABSTRACT ..................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGMENTS ............................................................................................... v LIST OF TABLES ........................................................................................................ viii LIST OF FIGURES ........................................................................................................ ix CHAPTER I. INTRODUCTION ......................................................................................... 1 Arthritis .................................................................................................... 1 Chondroitin Sulfate .................................................................................. 7 II. DETERMINATION OF MOLECULAR WEIGHTS AND DISACCHARIDE COMPOSITION OF CHONDROITIN SULFATE SAMPLES.............................................................................................. 17 Determination of Molecular Weight ...................................................... 18 Determination of Disaccharide Composition......................................... 20 III. DETERMINATION OF IN-VITRO ABSORPTION OF CS SAMPLES AND ITS RELATIONSHIP WITH MOLECULAR WEIGHT AND SULFATION ......................................................................................... 24 Introduction ............................................................................................. 24 Materials and Methods ............................................................................ 28 Results ..................................................................................................... 36 IV. DETERMINATION OF IN-VITRO ANTI-INFLAMMATORY ACTIVITY AND ITS RELATIONSHIP WITH MOLECULAR WEIGHT AND SULFATION OF CS SAMPLES ................................ 43 Introduction ............................................................................................ 43 vi Table of Contents (Continued) Page Materials and Methods ........................................................................... 46 Results .................................................................................................... 51 V. DISCUSSION .............................................................................................. 64 APPENDICES ............................................................................................................... 71 1: Materials and methods for the determination of molecular weights of CS samples ........................................................................................ 72 2: Materials and methods for the determination of disaccharide compositions of CS samples .................................................................. 75 3: Protocol for TNF-α ELISA .......................................................................... 78 4: Protocol for IL-1β ELISA ............................................................................ 80 5: Protocol for IL-6 ELISA .............................................................................. 82 6: Protocol for nitrite estimation ...................................................................... 85 REFERENCES .............................................................................................................. 86 vii LIST OF TABLES Table Page 1.1 Characteristics of CS from different sources ........................................................... 10 2.1 Assigned names, sources, and suppliers of CS samples used .................................. 17 2.2 Calculated molecular weights of CS samples .......................................................... 20 2.3 Disaccharide composition of CS samples (n=4) ...................................................... 22 3.1 Characteristics of parental Caco-2 cells ................................................................... 27 -6 -1 3.2 Ranking of samples based on Peff x 10 cm. s ....................................................... 39 4.1 Ranking of CS samples (conc. 5 μg/ml) according to TNF-α concentration ..................................................................................................... 52 4.2 Ranking of CS samples (conc. 15 μg/ml) according to TNF-α concentration .....................................................................................................